Epigenetics is a field of genetic or molecular biological research aimed at elucidation of the mechanisms of gene expression control that are not defined in terms of the genome. A major molecular mechanism that supports epigenetics is DNA methylation. DNA methylation is chemically stable, and such information is transmitted through cell division without changing the DNA sequence. Because of such characteristics, DNA methylation is considered to play a key role in various phenomena, such as tissue-specific gene expression, imprinting, X chromosome inactivation, and carcinogenesis. In recent years, many congenital or acquired diseases resulting from abnormality of the DNA methyltransferase gene or DNA methylation status, abnormality of DNA methylation in cloned animals, and the like have already been identified.
There have heretofore been a variety of methods aimed at analysis of DNA methylation. A variety of methods for analyzing DNA methylation have distinctive characteristics in terms of, for example, analysis sensitivity, completeness, resolution, and quantitative performance, and an adequate method is selected in accordance with the intended purpose. When the transcriptional repression mechanism of a specific gene is to be identified, for example, resolution at the single nucleotide level is necessary. When methylation frequency is to be analyzed, it is necessary to take quantitative performance and accuracy into consideration.
When detecting methylated DNA in a specific region, a nucleotide substitution reaction is carried out via bisulfite treatment to determine the DNA sequence. According to the nucleotide substitution reaction, unmethylated cytosine reacts with sodium bisulfite and it is converted into uracil. Since methylated cytosine does not react with sodium bisulfite, the methylation states of all cytosines can be detected as nucleotide differences in principle. Examples of conventional detection methods include a methylation-specific PCR (MSP) method of performing PCR, a real-time MSP method of performing quantitative PCR, bisulfite sequencing of performing TA cloning, the MassARRAY method of performing mass analysis, and pyrosequencing using a next-generation sequencer. In addition, the ICON-prove method, which does not require the bisulfite reaction, has been developed.
It is difficult to determine the expression levels of target proteins or microRNA based only on individual CpG methylation levels. Methylation patterns or continuity in such specific regions are important. While a variety of methods for detecting CpG methylation levels have been developed, methods for detecting CpG methylation patterns or continuity are limited to the bisulfite sequencing method.
The bisulfite-DGGE method was developed by P. Guldberg et al. (Non-Patent Document 2) based on the denaturing gradient gel electrophoresis (DGGE) method proposed by Abrams and Stanton (Non-Patent Document 1). According to the bisulfite-DGGE method, the sample after bisulfite treatment is amplified via PCR, and the amplicons thereof are then subjected to electrophoresis using a gel consisting of polyacrylamide and a denaturing agent having density gradient. Separation is carried out based on differences in the double-stranded DNA molecular weights in a polyacrylamide-density-gradient-dependent manner, separation is further carried out based on differences in the degrees of double-stranded DNA denaturation in a denaturing-density gradient-dependent manner, and separation based on differences between uracil (unmethylated cytosine) and cytosine (methylated cytosine) in the target region is then carried out. According to such method, visual evaluation can be performed, as with the case of the MSP method, and a band of a gel after electrophoresis can be used for sequencing. Compared with the bisulfite sequencing method, the bisulfite-DGGE method can be completed within a remarkably short period of time.
According to the bisulfite-DGGE method, however, PCR amplification is carried out after bisulfite treatment. This causes conversion of unmethylated cytosine into thymine, the resulting thymine is mixed with thymine that is originally present in the DNA sequence, and similar sequences then increase. Such increase often results in misannealing in PCR. When the number of PCR cycles is increased, accordingly, non-specific amplicons are disadvantageously amplified. When trace amounts of samples are to be analyzed or minor DNA methylation patterns are to be detected, it is necessary to increase the number of PCR cycles, and the resolution consequently deteriorates according to the bisulfite-DGGE method. Also, the bisulfite-DGGE method requires density gradients of both polyacrylamide and a denaturing agent, when preparing an electrophoresis gel. When the target is changed, accordingly, gel optimization becomes necessary, and such necessity disadvantageously complicates the procedure.